Plasmonic nanohole array biosensor for label-free and real-time analysis of live cell secretion

Cell secretion dynamics plays a central role in physiological and disease processes. Due to its various temporal profiles, it is essential to implement a precise detection scheme for continuous monitoring of secretion in real time. The current fluorescent and colorimetric approaches hinder such applications due to their multiple time-consuming steps, molecular labeling, and especially the 'snapshot' endpoint readouts. Here, we develop a nanoplasmonic biosensor for real-time monitoring of live cell cytokine secretion in a label-free configuration. Our nanoplasmonic biosensor is composed of gold nanohole arrays supporting extraordinary optical transmission (EOT), which enables sensitive and high-throughput analysis of biomolecules. The nanobiosensor is integrated with an adjustable microfluidic cell module for the analysis of live cells under well-controlled culture conditions. We achieved an outstanding sensitivity for the detection of vascular endothelial growth factor (VEGF) directly in complex cell media. Significantly, the secretion dynamics from live cancer cells were monitored and quantified for 10 hours while preserving good cell viability. This novel approach of probing cytokine secretion activity is compatible with conventional inverted microscopes found in a common biology laboratory. With its simple optical set-up and label-free detection configuration, we anticipate our nanoplasmonic biosensor to be a powerful tool as a lab-on-chip device to analyze cellular activities for fundamental cell research and biotechnologies.

Optofluidic nanoplasmonic biosensor for label-free live cell analysis in real time

Hatice Altug, Alexander Belushkin, Xiaokang Li, Maria Soler Aznar, Filiz Yesilköy

Cell signaling activities play a critical role in physiological and disease processes. The analysis of the tumor microenvironment or the immune system activation is nowadays providing valuable insights towards disease understanding and novel therapies development. Due to the various dynamic profiles, it is essential to implement a continuous monitoring methodology for accurate analysis. The current fluorescent and colorimetric approaches hinder such applications due to their multiple time-consuming steps, molecular labeling, and the ‘snapshot’ endpoint readouts. Photonics technology, and especially nanoplasmonic biosensors offer a unique opportunity to implement lab-on-a-chip systems that provide highly sensitive and label-free analysis of cell signaling events in real time. Here, we will present a microfluidics-integrated nanoplasmonic biosensor for long-term and real-time monitoring of cell secretion activity. The biosensor consists of a gold nanohole array supporting extraordinary optical transmission (EOT), which has been optimized to enable ultra-sensitive and high-throughput biomolecular detection. The nanobiosensor is integrated with a specifically designed microfluidic system that provides well-controlled cell culture conditions for long-term monitoring. We achieved an outstanding sensitivity for the detection of vascular endothelial growth factor (VEGF) directly secreted from microfluidic-cultured cancer cells. We demonstrated real-time monitoring for over 10 hours, preserving good cell viability. The multiplexing capability of our nanobiosensor could enable simultaneous analysis of different cell types and molecules-of-interest. Thus, our innovative approach of probing live cells can be a powerful tool to evaluate cellular activities for diagnostics and novel therapy development.

2018

Multiplexed nanoplasmonic biosensor for one-step simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae in urine

Hatice Altug, Alexander Belushkin, Andrea Cavallini, Maria Soler Aznar

Development of rapid and multiplexed diagnostic tools is a top priority to address the current epidemic problem of sexually transmitted diseases. Here we introduce a novel nanoplasmonic biosensor for simultaneous detection of the two most common bacterial infections: Chlamydia trachomatis and Neisseria gonorrhoeae. Our plasmonic microarray is composed of gold nanohole sensor arrays that exhibit the extraordinary optical transmission (EOT), providing highly sensitive analysis in a label-free configuration. The integration in a microfluidic system and the precise immobilization of specific antibodies on the individual sensor arrays allow for selective detection and quantification of the bacteria in real-time. We achieved outstanding sensitivities for direct immunoassay of urine samples, with a limit of detection of 300 colony forming units (CFU)/mL for C. trachomatis and 1500 CFU/mL for N. gonorrhoeae. The multiplexing capability of our biosensor was demonstrated by analyzing different urine samples spiked with either C trachomatis or N. gonorrhoeae, and also containing both bacteria. We could successfully detect, identify and quantify the levels of the two bacteria in a one-step assay, without the need for DNA extraction or amplification techniques. This work opens up new possibilities for the implementation of point-of-care biosensors that enable fast, simple and efficient diagnosis of sexually transmitted infections.

2017

Towards a point-of-care nanoplasmonic biosensor for rapid and multiplexed detection of pathogenic infections

Hatice Altug, Alexander Belushkin, Xiaokang Li, Maria Soler Aznar, Filiz Yesilköy

The implementation of multiplexed point-of-care biosensors is a top priority to address the current epidemic problems originated by widespread pathogenic infections, like those caused by viruses or bacteria. A rapid and accurate detection, identification, and quantification of the infectious pathogens is essential not only to facilitate a prompt treatment but also to prevent onward transmission, reduce economic expenses, and significantly promote healthcare in resource-constrained environments. We have developed a nanoplasmonic biosensor based on nanohole arrays for fast and highly sensitive analysis in a simple and direct configuration. Our microarray is integrated into a microfluidic system to allow for high-throughput detection of multiple targets in a few minutes, without the need of sample pretreatment or amplification steps. Previously, we demonstrated the utility of the biosensor for the detection of hazardous live viruses, such as the Ebola or Vaccinia viruses, measured directly in biological media. Most recently we proved the truly multiplexing capability of our plasmonic microarray with the simultaneous identification and quantification of Chlamydia trachomatis and Neisseria gonorrhoeae in urine samples. We are able to detect and distinguish the two different bacteria with detection limits in the range of 10(2)-10(3) bacteria/mL. With recent advances in plasmonics, optimized surface chemistry, and microfluidics integration, our biosensors could provide a non-invasive and rapid diagnosis at the point of care, especially when we combine the detection on a compact and low-cost optical reader.

SPIE-INT SOC OPTICAL ENGINEERING2018